WO2020140530A1 - Pseudopeptide, preparation method therefor and uses thereof - Google Patents

Abstract

Provided are a pseudopeptide, which is a compound of formula I or a stereoisomer thereof, a mixture of stereoisomer or a pharmaceutically acceptable salt thereof. The N-R₄, N-R₃, N-R₂ and N-R₁ of formula (I) represent respectively a pseudopeptide (II) showing a strong ability to bind to an EpCAM protein and can be used as molecular probe for diseases associated with EpCAM protein. The pseudopeptide is low cost, and is used for efficiently and accurately treating diseases associated with EpCAM protein through targeted therapy, in vivo imaging and prognostic prediction.

Description

Peptide, preparation method and use thereof

This application claims the priority of China Patent Application No. 201910005411.2 filed on January 3, 2019, and the contents of the above-mentioned Chinese Patent Application Publication are cited in its entirety as part of this application.

Technical field

The present disclosure relates to the field of biomedical technology. Specifically, the embodiments of the present disclosure relate to a peptoid, its preparation method and use.

Background technique

Epithelial specific adhesion molecule (Ephelhelial cell adhesion molecule, EpCAM) belongs to the family of adhesion molecules, which is a single transmembrane protein encoded by the tumor-associated calcium signal transduction 1 (TACSTD1) gene and participates in regulation Cell adhesion between cells mediates functions such as signal transduction, cell migration, proliferation and differentiation. Under pathological conditions, EpCAM is expressed in almost all adenocarcinomas, including colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer and retinoblastoma. EpCAM activates the expression of c-myc gene, cyclin A/E and other proto-oncogenes by participating in the Wnt cascade reaction dependent on β-catenin, and has a tumorigenic effect. At the same time, EpCAM is also an important indicator of tumor prognosis. For example, under normal circumstances, EpCAM is negatively expressed in esophageal squamous cell epithelium. In primary esophageal squamous cell carcinoma, almost 80% of tumors have different levels of EpCAM expression. EpCAM The average interval of recurrence of strong positive esophageal squamous cell carcinoma after surgery was 9 months, and the average interval of postoperative relapse of negative, weak positive and positive EpCAM was 43 months, indicating that the overexpression of EpCAM affects the prognosis of esophageal squamous cell carcinoma. In addition, the overexpression of EpCAM such as breast cancer and gastric cancer is also an important indicator of cancer cell metastasis.

Cancer targeted therapy and in vivo imaging have become one of the hottest topics in academia and clinical medicine today. Traditionally, the antitumor drugs currently used clinically are mainly cytotoxic chemotherapy drugs, but these drugs have poor selectivity, large toxic and side effects, and are likely to cause adverse reactions. Although protein drugs such as antibodies have high specificity and low toxic and side effects, due to their large molecular mass, complex structure, easy to cause immune reactions and drug resistance, and the preparation process is so complicated that the price is high, it is difficult for ordinary cancer patients Suffered.

With the help of the high permeability and retention effect (EPR) of solid tumors, the new drug carrier can carry drugs to the tumor site through passive targeting. Specific targeted drug delivery requires the use of specific recognition elements to enable selective active enrichment of the drug at the tumor site, resulting in increased drug concentration and increased penetration, thereby significantly improving the efficacy. In vivo imaging uses identification elements as the main body, combined with fluorescence, nuclear magnetic imaging and other imaging methods to achieve the goal. Therefore, the recognition element is the top priority of the entire targeted drug and in vivo imaging. Among them, the recognition element includes antibody, polypeptide/peptoid and nucleic acid aptamer and other targeted molecular probes of specific receptor proteins in tumor sites. Among various targeting molecules, aptamer small molecules have a series of unique advantages, such as low immunogenicity, good tissue permeability, small molecular weight, high stability, easy modification and economical.

Therefore, it is desired to develop a peptoid molecular probe for diseases related to EpCAM protein, to achieve targeted treatment, in vivo imaging diagnosis and prognostic monitoring of diseases related to EpCAM protein at low cost, with precision and efficiency.

Summary of the invention

At least one embodiment of the present disclosure provides a peptoid, which is a compound of Formula I or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof,

NR ₄ , NR ₃ , NR ₂ and NR ₁ in formula I are respectively

For example, in the peptoids, pharmaceutically acceptable salts are hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, propionate, fumarate, ethanol Salt, pyruvate, malate, malonate, benzoate, cinnamate, mandelate, salicylate, maleate, citrate, succinate, tartrate, Mesylate, ethanesulfonate, or p-toluenesulfonate.

In one embodiment, the peptoid is a compound of formula I.

At least one embodiment of the present disclosure also provides a method for preparing a peptoid. The method includes the following steps:

(1) The compound of formula II undergoes an amidation reaction with the amino group at the end of the solid phase carrier resin to form an amide bond;

Among them, R is OH or Cl;

(2) Add a subunit to replace the bromine atom through nucleophilic substitution reaction;

(3) Repeat steps (1) and (2) until the synthesis of all subunits is completed,

Wherein, the subunit input sequence is: cysteine, mono-protected tetramethylene diamine, mono-protected tetramethylene diamine, alanine, ethanolamine, mono-protected tetramethylene diamine Isobutylamine, the single protection means that one amino group in the diamine is protected by an amino protecting group;

(4) The amino protecting group of the side chain is removed to cleave the peptoid from the resin.

For example, the cysteine used is L-cysteine, D-cysteine, or a mixture of both.

At least one embodiment of the present disclosure provides the use of any of the above-mentioned peptides in the preparation of a medicament for targeted treatment of diseases related to EpCAM protein.

In one embodiment, the disease associated with EpCAM protein is adenocarcinoma. For example, the disease associated with EpCAM protein is colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma, or primary esophageal squamous cell carcinoma .

At least one embodiment of the present disclosure further provides a pharmaceutical composition, which includes: any one of the above-mentioned peptides; and pharmaceutically acceptable excipients.

For example, the auxiliary material is any combination of one or more of excipients, diluents, carriers, flavoring agents, binders, and fillers.

At least one embodiment of the present disclosure also provides use of any one of the above pharmaceutical compositions for imaging detection or prognosis monitoring of diseases related to EpCAM protein.

In one embodiment, the disease associated with EpCAM protein is adenocarcinoma. For example, the disease associated with EpCAM protein is colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma, or primary esophageal squamous cell carcinoma .

At least one embodiment of the present disclosure also provides a chip including any of the above-mentioned peptides. For example, the peptoid is coupled to the surface of the chip. In one embodiment, the chip is a microfluidic chip.

At least one embodiment of the present disclosure also provides a kit for identifying circulating tumor cells, including: a box body, a microfluidic chip disposed in the box body, and a fluorescent probe disposed in the box body. The fluorescent probe is the above-mentioned peptoid with a fluorescent label.

BRIEF DESCRIPTION

In order to more clearly explain the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly described below. Obviously, the drawings in the following description only relate to some embodiments of the present invention, rather than limit the present invention. .

1 is a graph showing the results of cell-level imaging using a fluorescent-labeled molecular probe with the peptide of Example 1; and

2 is a graph showing the results of surface plasmon resonance imaging of the peptides of Example 1 bound to EpCAM proteins at concentrations of 5.68 nM, 11.4 nM, 22.8 nM, 45.6 nM, and 91.2 nM.

detailed description

The various documents and publications mentioned in this disclosure are hereby incorporated by reference. Unless otherwise defined, the technical or scientific terms used in the present disclosure shall have the usual meanings understood by persons of ordinary skill in the field to which the invention belongs.

The term "pharmaceutically acceptable" in this disclosure indicates that the substance or composition is chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or the mammal treated with it. "Pharmaceutically acceptable salt" in this disclosure refers to a pharmaceutically acceptable salt formed by the compound.

Similar words such as "include" or "include" used in this disclosure mean that the elements appearing before the word cover the elements listed after the word and their equivalents, but do not exclude other elements. The sub-unit (monomer) of the present disclosure refers to a raw material amine added to a solid-phase synthetic peptide. The subunit of the present disclosure refers to a structural unit that constitutes a peptoid.

Peptoids are peptide mimetics with N-substituted glycine as the structural unit. Compared with peptides, the side chain of peptoids is transferred from α-carbon to N. Unlike traditional peptides, which have only 20 amino acids, peptoids are synthesized by subunit synthesis. Its constituent units are determined by different amines. There are thousands of amines, so the sequence of peptoids is extremely rich. Different chemical sequence structures can be developed for different targets. And because peptoids are not recognized by enzymes, peptoids can effectively resist proteolysis in the body, which makes peptoids have more obvious advantages as molecular probes.

The drug delivery system and molecular imaging system developed based on peptoid molecular probes may enhance the stability of the drug, may improve the interaction of the drug with tumor cells and tissues, and may increase the circulating metabolic cycle in the body. Therefore, in vivo imaging diagnosis and enhancement of the drug It has advantages in terms of effectiveness, overcoming drug resistance and reducing toxic and side effects.

The present disclosure provides a peptoid, which is a compound of formula I or a stereoisomer, a mixture of stereoisomers, or a pharmaceutically acceptable salt thereof,

NR ₄ , NR ₃ , NR ₂ and NR ₁ in formula I are respectively

The term "stereoisomer" belongs to a type of isomers, and refers to isomers caused by the same order of interconnection of atoms or atomic groups in a molecule, but different spatial arrangements.

Regarding stereoisomers, the compound of formula (I) has one asymmetric carbon atom, which can exist as a single enantiomer, or as a mixture of enantiomers such as a racemic mixture or an enantiomer A mixture enriched by the structure exists. The present invention encompasses any stereoisomers including compounds of formula (I) or pharmaceutically acceptable salts thereof and mixtures of various forms thereof.

The peptoids of the present disclosure can prepare individual enantiomers or racemic mixtures by selecting raw materials. For example, in the preparation of a peptoid of the present disclosure, the cysteine raw material may be selected from a mixture of D-cysteine and L-cysteine.

The peptoids of the present disclosure can also be resolved by chiral resolution, for example by chiral HPLC, to obtain individual enantiomers.

In one embodiment of the present disclosure, the peptoid is a compound of formula I.

The peptoids of the present disclosure may exist in the form of salts. For example, the salt can be prepared by reacting a peptoid compound with an inorganic acid or an organic acid. The inorganic acid is, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. The organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, almond Acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

In one embodiment of the present disclosure, the salt is a pharmaceutically acceptable salt. For example, the pharmaceutically acceptable salt may be hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, propionate, fumarate, glycolate, pyruvate, Malate, malonate, benzoate, cinnamate, mandelate, salicylate, maleate, citrate, succinate, tartrate, methanesulfonate, ethanesulfonate Acid salt, or p-toluenesulfonate.

In an embodiment of the present disclosure, the pharmaceutically acceptable salt is, for example, hydrochloride, nitrate, sulfate, phosphate, formate, acetate, fumarate, maleate, lemon Acid salt, succinate, tartrate, mesylate, or p-toluenesulfonate.

The present disclosure also provides a method for preparing the above-mentioned peptoid. The peptoids of the present disclosure can be synthesized by solid phase synthesis according to the subunit sequence of Formula I. The solid-phase synthesis method is well known to those skilled in the art. For example, J. Am. Chem. Soc. 1992, 114, 10646-10647 reports a method for solid-phase synthesis of peptoids. The peptoids of the present disclosure can be synthesized by the solid phase synthesis method shown below.

In the above reaction formula 1, bromoacetic acid may be replaced with bromoacetyl chloride.

At least one embodiment of the present disclosure also provides a method for preparing a peptoid. The preparation method includes the following steps:

(1) The compound of formula II undergoes an amidation reaction with the amino group at the end of the solid phase carrier resin to form an amide bond;

Among them, R is OH or Cl;

(2) Add a subunit to replace the bromine atom through nucleophilic substitution reaction;

(3) Repeat steps (1) and (2) until the synthesis of all subunits is completed,

Wherein, the subunit input sequence is: cysteine, mono-protected tetramethylene diamine, mono-protected tetramethylene diamine, alanine, ethanolamine, mono-protected tetramethylene diamine Isobutylamine, the single protection means that one amino group in the diamine is protected by an amino protecting group;

(4) The amino protecting group of the side chain is removed to cleave the peptoid from the resin.

In a method for preparing peptoids according to an embodiment of the present disclosure, the cysteine subunit may be selected from a mixture of D-cysteine and L-cysteine. For example, the cysteine subunit can be selected from L-cysteine.

In the preparation method of the peptoids of the present disclosure, first, an acylation reaction between the compound of formula II and the amino group at the end of the solid phase carrier resin to form an amide bond occurs, and then the subsequent nucleophilic substitution reaction of the subunit is performed, and the formula is used again The compound II undergoes an amidation reaction to form an amide bond with the amino group of the previous subunit and a nucleophilic substitution reaction of the subsequent subunit until the synthesis of all subunits is completed.

As the amino protecting group, an amino protecting group known in the art for the synthesis of proteins, peptides or peptoids can be used without limitation, for example, in "Greene's Protective Groups in Organic Synthesis" 5th Edition by Peter GMWuts Listed amino protecting groups. In some embodiments, the amino protecting group is 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc). For example, in some embodiments, the amino protecting group is tert-butoxycarbonyl.

The reaction conditions for the amidation reaction in the above step (1) are not particularly limited, and conventional conditions for amidation reactions for protein, polypeptide or peptoid synthesis in the art can be used as long as they can acylate the amino group without destroying the class The function of the peptide is sufficient. For example, the above amidation reaction can be carried out in the presence of a condensing agent. The condensing agent may use, without limitation, a condensing agent known in the art for protein, polypeptide or peptoid synthesis. For example, the condensing agent may be a carbodiimide-based condensing agent, such as N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), etc.; benzotriazole condensing agent, such as 1-hydroxy-benzo-triazole (HOBt); benzene Sulfonyl chloride-based condensing agents, such as triisopropylbenzenesulfonyl chloride (TPS), etc.; succinimide-based condensing agents, such as disuccinimide carbonyl ester (DSC), succinimide diphenyl phosphate (SDPP), etc.; 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ); 3-(diethoxyphosphoryl)-oxy-1,2,3-benzotriazine-4 (3H)-one (DEPBT) and so on.

The reaction conditions of the nucleophilic substitution reaction in the above step (2) are not particularly limited, and conventional conditions for carrying out the nucleophilic substitution reaction of protein, polypeptide or peptoid synthesis in the art can be adopted as long as the bromine atom can be replaced and the The function of the peptoid may be destroyed, for example, it may be reacted at a temperature of 35 to 40°C for 30 minutes or more, 60 minutes or more, or 90 minutes or more.

In the above step (4), the removal of the side-chain amino protecting group and the cleavage of the peptoid from the resin can be performed simultaneously or successively. For example, first cleave the peptoid from the resin, and then remove the side chain amino protecting group; you can also remove the side chain amino protecting group first, and then cleave the peptoid from the resin; or remove the side chain amino group While protecting the group, the peptoid is cleaved from the resin. The removal of the side chain amino protecting group and the cleavage of the peptoid from the resin can use conventional conditions for protein, polypeptide or peptoid synthesis in the art, as long as the purpose can be achieved without destroying the function of the peptoid can. In one embodiment, a cleavage solution containing 95% trifluoroacetic acid, 2.5% ultrapure water, and 2.5% triisopropylsilane in volume ratio can be used to remove the peptoid from the side chain amino protecting group. Cracked down on the resin.

In the method for preparing a peptoid of the present disclosure, a step of purifying the obtained product may be included as necessary. The purification method is not particularly limited, and methods known in the art for purifying corresponding similar products, such as precipitation, filtration, dialysis, gel permeation chromatography, and HPLC, can be used.

The present disclosure also provides a drug for targeted treatment of diseases related to EpCAM protein. The peptoids of the present disclosure can specifically recognize EpCAM protein, therefore, they can be used as molecular probes for specifically recognizing EpCAM protein, and can be used to prepare drugs for targeted treatment of diseases related to EpCAM protein. In one embodiment, the disease associated with EpCAM protein is adenocarcinoma. For example, the disease associated with EpCAM protein is colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma, or primary esophageal squamous cell carcinoma .

The present disclosure also provides a pharmaceutical composition comprising any of the above-mentioned peptides and pharmaceutically acceptable excipients. For example, the pharmaceutically acceptable auxiliary materials are any one or more of excipients, diluents, carriers, flavoring agents, binders, and fillers.

The peptoids of the present disclosure have a high affinity for EpCAM protein, so they can realize targeted drug loading and localized imaging by recognizing EpCAM protein, provide new options for the diagnosis and treatment of diseases with high expression of EpCAM, and also for sequencing and digital PCR The results provide evidence of in vivo data.

The present disclosure also provides the use of the above pharmaceutical composition for imaging detection or prognosis monitoring of diseases related to EpCAM protein. In one embodiment, the disease associated with EpCAM protein is adenocarcinoma. For example, the disease associated with EpCAM protein is colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma, or primary esophageal squamous cell carcinoma .

The present disclosure also provides a chip (for example, a microfluidic chip) including the above-mentioned peptoid. In one embodiment, the peptoid is coupled to the surface of the chip for capture and diagnosis of circulating tumor cells.

Circulating tumor cells (CTC, Circulating Tumor Cell) is a general term for various types of tumor cells present in peripheral blood. Due to spontaneous or diagnostic operations, they are shed from solid tumor lesions (primary foci, metastases), and most CTCs enter peripheral blood. Apoptosis occurs or is swallowed, a few can escape and develop into metastases, increasing the risk of death of patients with malignant tumors. The existence and number of CTCs are important indicators of cancer progression and metastasis. Detecting and tracking the number of CTCs in peripheral blood can help patients with early screening, efficacy monitoring, prognosis judgment, and recurrence prediction.

The detection technology for CTCs can predict the occurrence of early tumors, and can detect tumor metastasis during the treatment of patients with drugs. In addition, it can also guide the use of drugs for subsequent treatment. CTCs are derived from primary tumors or metastatic tumors. CTCs can enter the blood vessels after detaching from the basement membrane. Because the content of CTCs in the blood is extremely low, and its size is similar to the size of white blood cells, this makes it difficult for CTCs to be detected by liquid biopsy technology. However, the surface of CTCs carries proteins that are highly expressed specifically in related cancers.

The chip of the present disclosure can use various blank chips known in the prior art for CTC capture and detection and are prepared in accordance with conventional methods; or commercially available blanks that can be used for CTC capture and detection Chip preparation. For example, the chip may be prepared from a PlexArray HT 3D chip purchased from Plexera Bioscience of the United States.

Coupling of peptoids to the surface of the chip can be achieved by formulating peptoids into a sample of peptoid molecular probes and spotting them on the surface of the chip and then incubating.

The chip of the present disclosure can realize diseases related to EpCAM protein (such as colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, etc.) by combining the surface plasmon resonance imaging technology CTC capture and diagnosis of retinoblastoma or primary esophageal squamous cell carcinoma).

The present disclosure also provides a kit for identifying circulating tumor cells, including: a box body, a microfluidic chip disposed in the box body, and a fluorescent probe disposed in the box body, the fluorescent probe The above peptides with fluorescent labels.

For example, the microfluidic chip includes a microvalve control layer and a microvalve film layer. The microvalve control layer is provided with six holes penetrating the control layer and three gas channels. The three holes are sample loading holes, which are connected to the substrate for sample and reagent inflow and outflow; the remaining three holes are respectively connected to three gas channels , Used to inject gas and control the opening and closing of the microvalve. The microvalve membrane layer is provided with three holes penetrating the membrane layer, respectively corresponding to the three sampling holes of the microvalve control layer.

For example, the outer dimensions of the microvalve control layer and the microvalve film layer should match the substrate.

There is no particular limitation on the type of fluorescent group used for fluorescent labeling, as long as it can impart fluorescence to the peptoid after modification and can also achieve the basic function of the peptoid. Peptoids can be modified with one or more fluorescent groups. For example, one fluorescent group can be modified to obtain a single fluorescent labeled peptoid, or two fluorescent groups can be used to obtain a dual fluorescent labeled peptoid. In some embodiments, the fluorescent group may be selected from blue fluorescent dyes, near infrared fluorescent dyes, green fluorescent dyes, etc. without limitation, such as coumarin-containing fluorescent groups, anthracene-containing fluorescent groups, rhodamine-based fluorescent groups Group, phenanthroline fluorophore, naphthalene-containing fluorophore, fluorescein isothiocyanate, carboxyfluorescein (FAM), fluorescein thiocyanate (FITC), dansyl chloride (dansyl chloride), 2,4- Dinitrobenzene (Dnp), carboxyl rhodamine 110 (carbo-xyrhodamine 110), Texas Red (Texas Red), pentamethine cyanine dye (Cy5) and heptamethine cyanine dye (Cy7), etc.

For example, in addition to microfluidic chips and fluorescent probes, the detection system can also include a fluorescent microscope (fluorescence imaging system), image analysis software (analysis counting system), and a pump to form a complete system to complete the processing and circulation of blood samples Isolation and counting of tumor cells.

For example, a fluorescence microscope is used to detect whether cells in a micro-V-shaped positioning array are fluorescent, and perform full-coverage fluorescence imaging of the functional area to obtain a multi-channel fluorescence image.

For example, image processing software is used to analyze images acquired by a fluorescence microscope and obtain the corresponding number of CTCs. The software accurately calculates the size, area, aspect ratio, roundness and other parameters of the cells in the image, selects the CTCs that meet the requirements, and counts the CTCs. According to the algorithm, the localized cells can be screened and identified, and the circulating tumor cells that match the fluorescent characteristics of the label can be identified, counted, and the cell position reported and the cell image enlarged.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The SPRi instrument in the following embodiments is Plexera Kx5V2, Plexera Bioscience LLC, USA. The instrument is mainly equipped with a 660nm LED light source, a CCD image collector and a sensor chip with a microfluidic channel. The instrument displays the reflected light intensity at each monitoring point Change with time and record as SPR curve.

Unless otherwise specified, "EpCAM" herein refers to the full-length epithelial cell adhesion molecule.

Unless otherwise specified, "nM" refers to "nmol/L", "μM" refers to "μmol/L", and "mM" refers to "mmol/L".

Example 1 Preparation of Formula I Peptide

Peptoids are synthesized by solid phase synthesis, which includes the following steps:

(1) Immerse Rink amide AM resin (substitution level 0.3mmol/g, 200mg) in N,N-dimethylformamide (DMF), swell it for 15 minutes, then discharge the DMF solution;

(2) Deprotection: dispose 20% hexahydropyridine in DMF solution, immerse the resin in excess deprotection solution for 10 min, the Fmoc protecting group is removed, exposing the free amino group;

(3) Cleaning the resin: Washing with dichloromethane (DCM) and DMF alternately 3 times each;

(4) Add 2M bromoacetic acid DMF solution (2.5ml) and 3.2M N,N'-diisopropylcarbodiimide (DIC) DMF solution (2.5ml) to Rink amide AM resin at 37℃ Under the reaction for 30 min, the amino group at the end of the resin is acylated;

(5) Add 2M DMF solution of the subunit to be added (5ml) and react at 37°C for 90min. Replace the bromine atom by nucleophilic substitution reaction to complete the synthesis of a subunit;

(6) Cleaning resin: Use DCM and DMF to clean the resin alternately 3 times;

(7) Repeat steps (4)-(6) until the synthesis of the remaining subunits is completed; wherein, the subunit input sequence is: L-cysteine, mono-protected tetramethylene diamine, mono-protected tetramethylene Methyldiamine, alanine, ethanolamine, mono-protected tetramethylenediamine, isobutylamine.

(8) After the synthesis is completed, the side chain protecting group is removed, and the peptoid is cleaved from the resin by treatment with 95% trifluoroacetic acid, 2.5% ultrapure water, and 2.5% triisopropylsilane for 2 hours.

(9) After filtration, dilution and freeze-drying, the peptoid is purified by HPLC.

Example 2 Specific targeting experiment of peptide-like molecular probe EpCAM high expression cell line

The specific steps of using fluorescence-labeled probes with peptoid molecules for cell-level imaging are as follows:

(1) Cell culture and passage: the cell model was selected as Huh7 cell line with high expression of EpCAM protein and human embryonic kidney 293T cell line with low expression of EpCAM (Shanghai Enzyme Biotechnology Co., Ltd.), Huh7 and 293T cells were used with 10 RPMI1640 medium with 1% fetal bovine serum and 1% penicillin and streptomycin and DMEM/High glucose medium were cultured in a 37°C constant temperature incubator (5% CO ₂ );

(2) When the degree of cell fusion reaches more than 90%, passage the cells, suck the medium away with a pipette, wash away the residual medium and cell debris with PBS, and then add 0.25% of pancreatin 37 containing EDTA Digest at ℃ for 2min, add medium containing serum to stop the digestion, centrifuge at 1000rpm for 5min, cells gather at the bottom of the centrifuge tube, aspirate the medium, add new medium, aspirate the cells to make them evenly dispersed in the medium During the cell count;

(3) Inoculate Huh7 and 293T cells into a special dish for laser confocal microscopy at a rate of 1×10 ⁵ cells per dish for 12 hours;

(4) Remove the medium, then add 200 μL of medium containing peptoid molecular probes respectively, and incubate at 4°C for 30 min;

(5) Wash the cells 3 times with serum-free medium, then add 1 μg/mL DAPI to stain the nuclei for 15 min, and then wash them with PBS 3 times, then immediately observe and take a picture under a 100-fold oil microscope under a laser confocal microscope. All parameter settings for all sample microscopes are the same.

The test results are shown in Figure 1,

Left 1 indicates that after adding FITC-labeled peptoid probes to Huh7 cells, the peptoid probes target the EpCAM protein on the Huh7 cell membrane, so there is obvious FITC green fluorescence (red arrow) on the cell membrane.

Left 2 indicates that after adding the FITC-labeled peptoid probe to 293T cells, the EpCAM protein is not highly expressed on the 293T cell membrane, so no obvious FITC fluorescence signal is observed on the membrane, but FITC fluorescence is present in the solution (red arrow) , Due to the dilution effect of the solution, it is not strong)

Right 1 indicates that cells from left 1 stain the nucleus by DAPI. Right 2 indicates that the left 2 cells stained nuclei by DAPI.

Huh7 cells to which the peptide of Example 1 was added could observe obvious green fluorescence, while 293T cells in the control group had weak green fluorescence or no green fluorescence. Therefore, Figure 1 shows that peptoid-like molecular probes can specifically target EpCAM protein on the Huh7 membrane to form localization.

Example 3 The binding ability between peptide-like molecular probes and EpCAM protein

The specific steps for testing the binding ability between peptoid probe and EpCAM protein using surface plasmon resonance imaging technology are as follows:

(1) Dissolve the peptoid of Example 1 in ddH ₂ O at a concentration of 1 mM;

(2) Spot the samples of the above-mentioned peptoid molecular probes on the surface of a 3D chip (PlexArray HT, Plexera Bioscience, USA). Repeat 3 points for each sample. After incubating at 4°C for 12 hours, use 10X PBS, 1XPBS , Ultra-pure water is washed sequentially. Then, the chip was blocked with 1M hydrochloric acid aminoethanol for 30 minutes, then washed 5 times with ultrapure water, and finally dried with clean nitrogen;

(3) Install the chip on the SPRi instrument, measure the SPRi angle and adjust to the best optical position, select the relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL/s;

(4) Select PBS as the buffer and pass it into the flow cell until the baseline is stable, and then pass the EpCAM protein solutions with concentrations of 5.68 nM, 11.4nM, 22.8nM, 45.6nM, and 91.2nM for detection. The binding time is 300 seconds and the dissociation time For 300 seconds, phosphoric acid was passed between each concentration for regeneration.

The detection result is shown in Figure 2. The ordinate uRIU is the unit of the combined signal strength, which is obtained by AU conversion.

Fitted with BIA evaluation version 4.1 software (Biacore, Inc.), the equilibrium dissociation constant KD was 6.77×10 ^-8 mol/L, which indicates that the peptoid of Example 1 has a relatively high affinity with EpCAM.

In summary, the peptide-like molecular probe of the present invention is a molecular targeting probe with high sensitivity and high affinity for EpCAM protein, which provides a new method for the targeted treatment or imaging detection of cancers with high expression of EpCAM. select.

The present disclosure illustrates the process of the present invention through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to implement. In the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other to obtain a new embodiment.

Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of the selected raw materials of the present invention, addition of auxiliary components, choice of specific methods, etc., fall within the scope of protection and disclosure of the present invention. The protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (17)

1. A peptoid, which is a compound of formula I or its stereoisomers, a mixture of stereoisomers or their pharmaceutically acceptable salts,

   

   NR ₄ , NR ₃ , NR ₂ and NR ₁ in formula I are respectively

   
2. The peptoid according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, propionate, rich Maleate, glycolate, pyruvate, malate, malonate, benzoate, cinnamate, mandelate, salicylate, maleate, citrate, succinic acid Salt, tartrate, methanesulfonate, ethanesulfonate, or p-toluenesulfonate.
3. The peptoid according to claim 1, which is a compound of formula I.
4. Use of the peptoid according to any one of claims 1 to 3 in the preparation of a medicament for targeted treatment of diseases related to EpCAM protein.
5. The use according to claim 4, wherein the disease associated with EpCAM protein is adenocarcinoma.
6. The use according to claim 4, wherein the diseases related to EpCAM protein are colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma Cancer or primary squamous cell carcinoma of the esophagus.
7. A pharmaceutical composition comprising:

   The peptoid according to any one of claims 1-3; and

   Pharmaceutically acceptable excipients.
8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable excipient is any one or more of excipients, diluents, carriers, flavoring agents, binders, and fillers.
9. Use of the pharmaceutical composition according to claim 7 or 8 for imaging detection or prognosis monitoring of diseases related to EpCAM protein.
10. The use according to claim 9, wherein the disease associated with EpCAM protein is adenocarcinoma.
11. The use according to claim 9, wherein the diseases related to EpCAM protein are colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, stem cell cancer, retinoblastoma Cancer or primary squamous cell carcinoma of the esophagus.
12. A chip comprising the peptoid according to any one of claims 1-3.
13. The chip according to claim 12, wherein the peptoid is coupled to the surface of the chip.
14. The chip according to claim 12 or 13, wherein the chip is a microfluidic chip.
15. A kit for identifying circulating tumor cells, comprising: a box body, a microfluidic chip arranged in the box body, and a fluorescent probe arranged in the box body, the fluorescent probe is provided with fluorescence The labeled peptoid according to any one of claims 1-3.
16. A method for preparing the peptoid according to claim 1, comprising the following steps:

    (1) The compound of formula II undergoes an amidation reaction with the amino group at the end of the solid phase carrier resin to form an amide bond;

    

    Among them, R is OH or Cl;

    (2) Add a subunit to replace the bromine atom through nucleophilic substitution reaction;

    (3) Repeat steps (1) and (2) until the synthesis of all subunits is completed,

    Wherein, the subunit input sequence is: cysteine, mono-protected tetramethylene diamine, mono-protected tetramethylene diamine, alanine, ethanolamine, mono-protected tetramethylene diamine Isobutylamine, the single protection means that one amino group in the diamine is protected by an amino protecting group;

    (4) The amino protecting group of the side chain is removed to cleave the peptoid from the resin.
17. The production method according to claim 16, wherein the cysteine used is L-cysteine, D-cysteine or a mixture of both.

Images